Lipids, stored as triacylglycerol (TG), play an essential role as reserve of metabolic energy in insects. Utilization of fat body TG stores requires TG hydrolysis (lipolysis). Because fatty acids provide energy to other tissues as well as to the fat body cell, lipolysis must be coupled to multiple metabolic pathways. The long-term goal of this project is to understand the mechanism of regulation of lipolysis. During the previous period we identified two key components of the lipolytic pathway in insects. The main lipase found in the insect fat body, TG-lipase (TGL) homolog to Drosophila melanogaster CG8552, and a lipid droplet-associated protein, Lsd1, whose phosphorylation stimulates the rate of lipolysis. Lsd1 is one of the two PAT proteins, Lsd1 and Lsd2, of insect lipid droplets and plays a major role in the regulation of lipolysis. Lsd1 contains multiple conserved phosphorylation sites that regulate its activity. Lsd1 also has structurally conserved domains that are expected to play a role in its interaction with lipases and regulatory proteins and thus in the regulation of lipolysis. One of the goals of this project is to dissect the role of different regions of the Lsd1 molecule in the regulation of lipolysis. Other cytosolic and lipid droplet proteins, such as Lsd2, are expected to be involved in the regulation of lipolysis. This project also proposes to identify some of these proteins using a proteomic approach coupled to specific interaction assays and global studies to define differences in the protein composition of lipid droplets from several insects. Strong evidence indicating that the rate of lipolysis in the insect fat body is affected by the redox state of adipocytes led us to propose the investigation of this mechanism. Because the rate of TG hydrolysis must be modulated by signals originated from different pathways, we hypothesize that the redox state of the fat body cell, which is sensitive to multiple metabolic changes, plays a major role in the regulation of TG hydrolysis. In this project we will study the mechanism by which the redox state of the cell affects lipolysis. These studies will contribute to the understanding of the regulation of the utilization of energy stores in insects. This aspect of the biology of insects may be important to find mechanisms of control of insects that act as vectors of human diseases. PUBLIC HEALTH RELEVANCE: Energy metabolism and its regulation are key processes defining insect survival and reproduction. TGs are the predominant form of energy storage in all animals. The ability to store and release this energy involves a carefully regulated balance between TG synthesis and hydrolysis. Specialized mechanisms are expected in insects, including vectors of human diseases, to meet their unique metabolic needs, such as the capacity to mobilize massive amounts of lipid in a short period of time. These metabolic distinctions must be reflected in the molecular mechanisms involved in TG mobilization in insects. The long term goal of this project is to achieve a clear understanding of the regulation of triacylglycerol metabolism in insects.